Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene.

Human obesity has an inherited component, but in contrast to rodent obesity, precise genetic defects have yet to be defined. A mutation of carboxypeptidase E (CPE), an enzyme active in the processing and sorting of prohormones, causes obesity in the fat/fat mouse. We have previously described a women with extreme childhood obesity (Fig. 1), abnormal glucose homeostasis, hypogonadotrophic hypogonadism, hypocortisolism and elevated plasma proinsulin and pro-opiomelanocortin (POMC) concentrations but a very low insulin level, suggestive of a defective prohormone processing by the endopeptidase, prohormone convertase 1 (PC1; ref. 4). We now report this proband to be a compound heterozygote for mutations in PC1. Gly-->Arg483 prevents processing of proPC1 and leads to its retention in the endoplasmic reticulum (ER). A-->C+4 of the intro-5 donor splice site causes skipping of exon 5 leading to loss of 26 residues, a frameshift and creation of a premature stop codon within the catalytic domain. PC1 acts proximally to CPE in the pathway of post-translational processing of prohormones and neuropeptides. In view of the similarity between the proband and the fat/fat mouse phenotype, we infer that molecular defects in prohormone conversion may represent a generic mechanism for obesity, common to humans and rodents.

Association of the prohormone convertase 2 gene (PCSK2) on chromosome 20 with NIDDM in Japanese subjects.

Proinsulin is converted to insulin by the concerted action of two sequence-specific subtilisin-like proteases termed prohormone convertase 2 (PC2) and prohormone convertase 3. PC2 is a type II proinsulin-processing enzyme, and it cleaves the proinsulin molecule on the COOH-terminal side of dibasic peptide, Lys64-Arg65, which joins the C-peptide and the A-chain domains. We have previously cloned and characterized the exon-intron organization of the human PC2 gene (gene symbol PCSK2), localized this gene to human chromosome 20 band p11.2 by fluorescence in situ hybridization, and identified a simple tandem-repeat DNA polymorphism (STRP) in intron 2 of the form (CA)n, suitable for genetic studies. Since non-insulin-dependent diabetes mellitus (NIDDM) is associated with increased secretion of proinsulin and proinsulin-like molecules, we conducted a case-control study to determine whether a genetic variation in PCSK2 might contribute to the development of NIDDM. The study population consisted of 152 Japanese NIDDM subjects and 102 normal healthy nondiabetic control subjects matched for age and body mass index. The subjects were genotyped at the STRP in intron 2, and the results indicated a significant difference (P = 0.004) in the overall allele frequency distribution between the two groups. The A1 allele was found more frequently in NIDDM than in nondiabetic subjects (11 vs. 4%, P = 0.0068). The NIDDM patients were divided into two subgroups according to the presence or absence of the A1 allele.(ABSTRACT TRUNCATED AT 250 WORDS)

Is islet amyloid polypeptide a significant factor in pathogenesis or pathophysiology of diabetes?

Islet amyloid polypeptide (IAPP) or amylin, a recently discovered minor secretory peptide of the beta-cell related to calcitonin gene-related peptide (CGRP), is a constituent of amyloid deposits in the islets of many non-insulin-dependent (type II) diabetic individuals and some elderly nondiabetic subjects. IAPP is synthesized as a small precursor at a level of approximately 1% that of insulin and is processed, amidated, stored in beta-granules, and released along with insulin and C-peptide. Analysis of its gene (located on chromosome 12) supports an evolutionary relationship to calcitonin and CGRP, peptides with which it shares some biological actions. Like CGRP, IAPP antagonizes the action of insulin mainly at the level of muscle glycogen synthesis, but the levels required for this effect seem to be considerably higher than reported circulating levels. No evidence for overproduction of IAPP in diabetic subjects has been found thus far, but much more work is necessary to define its normal secretory rates and clearance. Other proposed actions of IAPP include serum calcium-lowering effects and smooth muscle relaxation; the latter effect might promote the uptake of insulin into the circulation within the islets. Deposition of amyloid is species selective due to structural differences within the central part of the molecule and may be initiated intracellularly in type II diabetes by several mechanisms. No differences in the structure of IAPP or its precursor have been found in individuals with maturity-onset diabetes of the young or type II diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Human prohormone convertase 3 gene: exon-intron organization and molecular scanning for mutations in Japanese subjects with NIDDM.

Proinsulin is converted to insulin by the concerted action of two sequence-specific subtilisin-like proteases termed prohormone convertase 2 (PC2) and prohormone convertase 3 (PC3). PC3 is a type I proinsulin-processing enzyme that initiates the sequential processing of proinsulin to insulin by cleaving the proinsulin molecule on the COOH-terminal side of the dibasic peptide, Arg31-Arg32, joining the B-chain and C-peptide. Thus, PC3 plays a key role in regulating insulin biosynthesis. Expressions of insulin and PC3, but not PC2, are coordinately regulated by glucose, consistent with the important role of PC3 in regulating proinsulin processing. NIDDM is associated with increased secretion of proinsulin and proinsulin-like molecules, suggesting that mutations in the PC3 gene may be involved in the development of this disorder. To examine this hypothesis, we have isolated and characterized the human PC3 gene and screened it for mutations in a group of Japanese subjects with NIDDM. The PC3 gene consists of 14 exons spanning more than 35 kb. The exon-intron organization of PC2 and PC3 genes are conserved, consistent with a common evolutionary origin for the prohormone convertase gene family. Single-strand conformational analysis and nucleotide sequencing of the entire coding region of the PC3 gene in 102 Japanese subjects with NIDDM revealed missense mutations in exons 2 (Arg/Gln53) and 14 (Gln/Glu638), neither of which was associated with NIDDM in this population. These data suggest that genetic variation in the PC3 gene is unlikely to be a major contributor to NIDDM susceptibility in Japanese.

Missense mutation of amylin gene (S20G) in Japanese NIDDM patients.

Many studies suggest that amylin, which is cosecreted with insulin from islet beta-cells, is a biologically active peptide and modulates plasma glucose levels. We therefore scanned the amylin gene for mutations in 294 Japanese NIDDM patients by single-strand conformational polymorphism, and we found a single heterozygous missense mutation (Ser-->Gly at position 20: S20G mutation) in 12 NIDDM patients (frequency 4.1%). None of the 187 nondiabetic subjects or 59 IDDM patients had the mutation. Of 12 patients carrying the mutation, 8 were diagnosed as having NIDDM at a relatively early age (< or = 35 years), and they had severe diabetes and strong family histories of late-onset NIDDM. On the other hand, the remaining four patients were diagnosed as having NIDDM after age 51, and they had mild diabetes without family histories of diabetes. In high-performance liquid chromatography analysis, a small amount (16%) of amylin immunoreactivity appeared in the position corresponding to normal amylin and a much larger amount (84%) appeared in the position corresponding to mutant amylin. These findings suggest that the S20G mutation of the amylin gene may play a partial role in the pathogenesis of early-onset NIDDM in the Japanese population and may also provide an important model to investigate the true physiological action of amylin.

A case of Albright's hereditary osteodystrophy-like syndrome complicated by several endocrinopathies: normal Gs alpha gene and chromosome 2q37.

We report a sporadic case of Albright's hereditary osteodystrophy (AHO)-like syndrome with several endocrinopathies. A 37-yr-old woman had an appearance of AHO but did not have renal PTH resistance. Her case was complicated by non-insulin-dependent diabetes mellitus with severe insulin resistance, central diabetes insipidus, and hyposecretion of GH. Most patients with AHO are found in a family of pseudohypoparathyroidism type-Ia and have a heterozygous mutation that inactivates the alpha-subunit of Gs (Gs alpha), the stimulatory regulator of adenylyl cyclase. Some sporadic cases occur in which patients with phenotype similar to AHO have a deletion of chromosome 2q37. However, in this patient, both the Gs alpha gene structure and the biological activity were normal. In addition, chromosome analysis revealed a normal pattern with no visible deletion of chromosome 2q37. Our findings suggest that one or more other factors may be involved in the pathogenesis of AHO-related disease.

Novel putative protein tyrosine phosphatases identified by the polymerase chain reaction.

Protein tyrosine phosphatases (PTPases) are a family of enzymes that specifically dephosphorylate phosphotyrosyl residues in selected protein substrates. To more fully understand the regulatory role of protein tyrosine phosphorylation and dephosphorylation in cellular signal transduction, characterization of PTPases is essential. Using the polymerase chain reaction and degenerate oligonucleotide primers corresponding to conserved amino acid sequences within the catalytic domain of PTPases, we have identified 11 PTPase-related human liver cDNA sequences. Five of these have not been described previously. These results indicate that, like protein tyrosine kinases, PTPases may also comprise a gene family with a large number of members.

Nucleotide sequence and analysis of the mouse SPC3 promoter region.

Insulin is converted from the higher molecular weight proprotein, proinsulin by highly specific proteolytic cleavage at two dibasic amino acid sites. SPC3 and SPC2, two recently identified prohormone convertase that are specifically expressed in beta cells and other neuroendocrine cells, appear to be responsible for those cleavages. We have sequenced the 5'-upstream region of the SPC3 gene and examined its promotor/enhancer activity and most of several deletion mutants in several cell lines. This region contains no CAAT box but has several non-functional TATA-like sequences and several putative transcriptional regulatory elements, including AP-1, Sp1 and cAMP response elements. These features are not unlike those of the human SPC2 upstream region. In beta TC3 insulinoma cells, the sequence between the EcoRI (620 bp) and NsiI (702 bp) sites seems to be important for gene expression, while the sequence between the NsiI and DraI (775 bp) sites may contain strong enhancer element(s).

Human islet amyloid polypeptide transgenic mice as a model of non-insulin-dependent diabetes mellitus (NIDDM).

To model islet amyloidogenesis in NIDDM and explore the glucoregulatory role of islet amyloid polypeptide (IAPP), we have created transgenic mice containing a rat insulin-I promoter-human IAPP fusion gene. Expression of human IAPP was localized to the islets of Langerhans, anterior pituitary and brain in transgenic animals; blood IAPP levels were elevated 5-fold while fasting glucose levels remained normal. Amyloid deposits have not been detected in transgenic islets suggesting that other co-existing abnormalities in NIDDM may be required for the formation of islet amyloid. These animals provide a unique model for exploring this hypothesis and other proposed functions of IAPP.

Cloning of mouse islet amyloid polypeptide gene and characterization of its promoter.

Islet amyloid polypeptide (IAPP) was isolated from islet amyloid deposits in patients with insulinoma and pancreatic islets of non-insulin-dependent diabetes mellitus (NIDDM) and several reports suggested that it may contribute to the development of NIDDM. IAPP is mainly expressed and synthesized in pancreatic B cells and cosecreted with insulin, so analysis of the transcriptional regulation of the IAPP gene would be helpful for the elucidation of pancreatic B cell specific gene expression. The mouse IAPP gene spans about 5.8 kb and, like the human and rat genes, it consists of three exons, and analysis of the promoter/enhancer activity of mouse IAPP gene reveals the region from -171 to -87 bp to be essential. Within this region, an E-box like sequence, CACCTG (-122 to -117 bp), and a TAAT-box like sequence, TTAATG (-139 to -134 bp), are thought to be important. The disruption of each sequence resulted in a severe decrease in promoter activity, although the decrease was less in the disruption of the E-box than that of TAAT-box like sequence, suggesting the latter is more important for IAPP gene transcription. Like the rat IAPP gene, the CCAAT-box, which does not exist in the human gene, was identified in the mouse gene, indicating the possibility of species difference in the IAPP gene transcriptional mechanism. An enhancer-like activity was also identified within intron 1, although further elucidation is necessary.

Restriction fragment length polymorphisms near the islet amyloid polypeptide gene in Japanese subjects.

Two restriction fragment length polymorphisms (RFLPs) near the human islet amyloid polypeptide (IAPP) gene were examined in 50 Japanese patients with non-insulin-independent diabetes mellitus (NIDDM) and 54 non-diabetic controls. RFLPs were identified with the enzymes PvuII (A1 = 21 kb and A2 = 18 kb) and BglII (B1 = 9 kb and B2 = 7 kb). These RFLPs were in complete linkage disequilibrium with A1 which was in disequilibrium with B2, as was A2 with B1. Since these two RFLPs map to different locations in the 5'-flanking region of the IAPP gene, they are most likely due to changes in the sequence of the sites recognized by PvuII and BglII rather than to an insertion/deletion-type DNA polymorphism. There were no differences in the genotypic or allelic frequencies of these RFLPs between Japanese subjects with NIDDM and non-diabetic controls implying that these RFLPs do not play a major role in the development of NIDDM in this population.

Molecular biology of islet amyloid polypeptide.

We investigated the relationship between non-insulin-dependent diabetes mellitus (NIDDM) and islet amyloid polypeptide (IAPP) gene by restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR)-direct sequencing analysis. Endonuclease BglII and/or PvuII RFLP analysis revealed no positive correlation of IAPP gene with NIDDM. In PCR-direct sequencing of 25 NIDDM patients, no nucleotide sequence differences were found. These data do not support the view that IAPP plays an important role in the pathogenesis of NIDDM. cDNAs encoding cat, rat, mouse, guinea pig and degu IAPP precursors were also cloned, and comparison of these predicted amino acid sequences clarified the species difference, especially between amyloid-forming and non-amyloid-forming species. Amino acid residues 25-28 of mature IAPP might be responsible for their amyloidogeneity. The alternative splicing transcripts of guinea pig IAPP gene were identified by using PCR. If these types of transcripts are translated, N-terminal mutated IAPP might be produced and act as an antagonist. The signal peptide cleavage site of rat IAPP precursor was also identified by an in vitro translation and processing system.

[Acute abdomina pain as a presenting symptom of varicella-zoster virus infection in an allogeneic bone marrow transplant].

A 26-year old man was admitted because of acute abdominal pain. He had received an allogeneic bone marrow transplant (BMT) for aplastic anemia 6 months before. All physical, laboratory, roentgenographic, and ultrasonographic studies were performed but nondiagnostic. On the fourth hospital day the patient developed visual disturbance and on the following day skin eruption appeared. Laboratory findings revealed severe liver dysfunction. We diagnosed this case as varicella-zoster virus (VZV) infection with visceral dissemination. Antiviral therapy with acyclovir was initiated and abdominal pain markedly reduced and visual acuity was recovered after 4 days. In case of VZV infection, acute abdominal pain prior to skin eruptions is rare. However in such cases the patients are highly fatal due to visceral dissemination. Antiviral therapy begun before visceral dissemination of VZV is highly effective in preventing serious disease, whereas it is less effective after dissemination. We consider that early diagnosis and treatment of VZV infection is necessary for BMT recipients who are undergoing immunosuppressive therapy.

[Analysis of the gene encoding human PC2, a prohormone processing enzyme].

Prohormone Convertase 2 (PC2) is a specific endoprotease responsible for the processing of proinsulin to insulin. PC2 is expressed in pancreatic islets, pituitary and brain but is very low or absent in most other tissues, such as liver, spleen and kidney. To evaluate the regulated expression of the human PC2 gene we have analyzed its structure and characterized its promoter. The gene spans > 130 kilobase pairs and consists of 12 exons. Comparison with the structure of the gene encoding human furin, revealed a high degree of conservation of exon-intron junctions. The hPC2 gene was localized to chromosome 20, band p11.2. The promoter region of the PC2 gene is very G + C rich and contains six potential Sp1 binding sites but no TATA or CAAT box. Analysis of the level of chrolamphenicol acetyltransferase activity with several deletion mutants identified the region from -1100 to -539 from the translation start site as essential for the PC2 promoter activity.

Sequences of islet amyloid polypeptide precursors of an Old World monkey, the pig-tailed macaque (Macaca nemestrina), and the dog (Canis familiaris).

The 37-amino acid islet amyloid polypeptide represents the major protein component present in islet amyloid deposits. Although the presence of islet amyloid is a characteristic pathological feature of the islets of humans, monkeys and cats with Type 2 (non-insulin-dependent) diabetes mellitus, it is not found in the islets of diabetic rats, mice or dogs. To further explore the molecular basis for these species differences in amyloid deposition we have used a polymerase chain reaction based method to clone cDNAs encoding the monkey (Macaca nemestrina) and dog (Canis familiaris) islet amyloid polypeptide precursors. The predicted amino acid sequence of the monkey precursor is 96% identical to that of the human protein; differences include one replacement in the signal peptide and three in the islet amyloid polypeptide domain. The sequence of the dog precursor is most closely related to that of the cat protein (85% identity); the sequences of dog and cat islet amyloid polypeptide differ only at two positions and are identical in the region of amino acids 20-29, the region thought to be primarily responsible for amyloidogenesis. Thus, amino acid residues in addition to those at positions 20-29 may facilitate the aggregation of islet amyloid polypeptide. The presence of amyloid deposits in some dog pancreatic endocrine tumours suggests that the dog protein can be amyloidogenic, perhaps due to elevated expression of islet amyloid polypeptide by the tumours relative to normal islets.

Organization of the human carboxypeptidase E gene and molecular scanning for mutations in Japanese subjects with NIDDM or obesity.

Insulin is synthesized in the pancreatic beta cell as a larger precursor molecule proinsulin which is converted to insulin and C-peptide by the concerted action of prohormone convertase 2 (PC2), prohormone convertase 3 (PC3) and carboxypeptidase E (CPE). One of the features of non-insulin-dependent diabetes mellitus (NIDDM) is an elevation in the proinsulin level and/or proinsulin/insulin molar ratio suggesting that mutations in these three proinsulin processing enzymes might contribute to the development of NIDDM. The identification of a mutation in the CPE gene of the fat/fat mouse which leads to marked hyperproinsulinaemia and late-onset obesity and diabetes is consistent with a possible role for mutations in CPE in the development of diabetes and obesity in humans. In order to test this hypothesis, we have isolated and characterized the human CPE gene and screened it for mutations in a group of Japanese subjects with NIDDM and obesity. The human CPE gene consists of 9 exons spanning more than 60 kb. Primer extension analysis identified the transcriptional start site at -141 bp from the translational start site. Single strand conformational polymorphism analysis and nucleotide sequencing of the promoter and entire coding region of the CPE gene in 269 Japanese subjects with NIDDM, 28 nondiabetic obese subjects and 104 nonobese and nondiabetic controls revealed three nucleotide changes, a G-to-T substitution at nucleotide -53, a G-to-A substitution at nucleotide -144 (relative to start of transcription) in the promoter region and a silent G-to-A substitution in codon 219. None of the nucleotide substitutions were associated with NIDDM or obesity. Thus, genetic variation in the CPE gene does not appear to play a major role in the pathogenesis of NIDDM or obesity in Japanese subjects.

Characterization of the promoter of the mouse prohormone convertase PC2 gene.

Prohormone convertase 2 (PC2) is a member of a family of mammalian subtilisin-like endoproteases that are involved in the processing of prohormones, neuropeptides and many other precursor derived proteins. The expression of PC2 is restricted to neuroendocrine tissues such as pancreatic islets, the pituitary and the brain. To understand the regulation of the PC2 gene, we cloned and characterized the promoter region of the mouse PC2 gene. The transcriptional start site of the mouse PC2 gene is identical to that of the human. There is 79% identity in the sequences of the promoter regions between the mouse and human PC2 genes. The mouse PC2 gene, like the human, does not have a TATA-like motif in the region just upstream of the start of the transcription. Studies with promoter-reporter gene, chloramphenicol acetyltransferase (CAT), constructs showed that the region from -400 to -170 bp was necessary for high level expression of the mouse PC2 gene in the betaTC-3 insulinoma cells.

Simple tandem repeat DNA polymorphism in the human glycogen synthase gene is associated with NIDDM in Japanese subjects.

We investigated the possible association between alleles of a simple tandem repeat DNA polymorphism in the human glycogen synthase gene and non-obese non-insulin-dependent diabetes (NIDDM) in Japanese subjects. Nine alleles (-4G, -3G, -2G, -1G, 0G, 1G, 2G, 3G, and 4G) were identified in the study group of 164 patients with NIDDM and 115 non-diabetic subjects. The overall frequency distribution of the glycogen synthase gene alleles was significantly different between the two groups (p = 0.0316). The 2G allele was found more frequently in patients with NIDDM than in non-diabetic subjects (17.7% vs 8.7%, p = 0.0016). These results suggest that the 2G allele could be a genetic marker of NIDDM in Japanese subjects.